Human Immunodeficiency Virus (HIV-1) is a member of the retroviral family which contains a single-stranded RNA genome and is considered the major etiological agent involved in the development of acquired immunodeficiency syndrome or AIDS. The World Health Organization estimates that as of the end of 2007 over 42 million people worldwide are infected and this number is growing. There continues to be a significant need for new drugs and drug combinations to combat this disease. There are a number of potential steps in the life cycle of the HIV virus for intervention with potential therapeutics. [1] A great deal of effort to develop drugs against HIV has been centered around HIV reverse transcriptase (RT), HIV protease, and more recently viral entry. The drugs that target HIV RT are divided into two general classes classes: nucleoside inhibitors (NRTIs) and non-nucleoside inhibitors (NNRTIs). The NRTI and NNRTI drugs currently approved by the FDA are summarized in FIGS. 3 & 4. [2-12]
The fully functional HIV reverse transcriptase has been shown to be a heterodimer containing two subunits-p66 and p51. Several structures are now available. The most recent ones are the 3-dimensional structure of HIV-1 RT-template/primer-dNTP poised for catalysis [13-16]. These structures will help guide our mechanistic and inhibitor design studies and help corroborate our functional studies. The structure of the HIV-1 RT catalytic complex is shown in FIG. 1. The overall topology of the p66 subunit has been described as a right hand containing fingers, palm, and thumb domains in addition to a connection domain and the RNAseH domain. The polymerase active site lies in the palm of the hand which contains three acidic residues considered to be essential catalytic residues (D185, D186, and D110) based upon mutagenesis studies [17, 18]. In addition there is a connection domain that joins the carboxyl terminal domain (which contains the RNAse H cleavage site) to the right hand. The p51 subunit although completely homologous in sequence to the p66 subunit is strikingly different in structure. The DNA binds along a groove, stretching from the polymerase active site to the RNAseH active site. The DNA has an A-like conformation in the polymerase active site and transitions to a B-like structure 4-5 base pairs away from the active site. The A- to B-form transition in the DNA structure is accompanied by an overall bend of about 40° as previously observed [15]. The NRTI and NNRTI binding sites are noted.
Studies have allowed the present inventors to develop the kinetic model shown in Scheme 1 (FIG. 2) describing the mechanism of polymerization for HIV RT. This is an ordered mechanism with the DNA binding first followed by the nucleotide. The DNA, containing a specified number of bases (DNAn), binds tightly to the RT forming an E●DNA complex that is slow to dissociate. The binding of the dNTP follows. A two step binding process of the dNTP controls the selectivity involved in incorporating the correct nucleotide. The dNTP first binds to the E●DNAn complex in a loose conformational state that is largely controlled by the base-pairing free energy upon recognition of the correct dNTP over the incorrect dNTP [19]. The binding of the correct dNTP induces a rate-limiting protein conformational change designated by E*●DNAn●dNTP. The formation of this tight ternary complex allows the critical transition state to be reached leading to very rapid chemistry to produce the elongated DNAn+1. After nucleotide incorporation, the enzyme must return to the loose state in order to release the PPi and a translocation step to allow the next round of reaction [20-22]. Additional evidence for the two conformational states of the ternary enzyme, DNA, dNTP complex are provided by the kinetic studies with non-nucleoside inhibitors [23, 24]. The most recent crystal structures nicely corroborate the inventors' initial kinetic model. The rate-limiting conformational state of the protein induced by the binding of the correct dNTP was visualized in the structure of the catalytic complex by the fingers domain closing in toward the palm domain to form a very tight enzyme*-DNA-dNTP complex [14].
The NNRTI class of inhibitors of HIV-1 RT bear no structural resemblance to the nucleosides. Structures of some of the clinically significant non-nucleoside analogs are shown in FIG. 4. Structures of some of the clinically significant nucleoside analogs are shown in FIG. 3. Many types of structurally diverse compounds have been shown to be very potent and selective inhibitors of RT [25]. The 3-dimensional structures of a number of these analogs bound to the RT enzyme have shown that they bind in a hydrophobic pocket proximal to the active site [25]. The structural studies have shown that a common structure feature of these compounds is their ability to adopt a “butterfly” type shape in this hydrophobic pocket. For successful replication of the viral genome, RT must catalyze a complex series of biochemical reactions in which the single-stranded RNA is converted into a double-stranded DNA copy that will be integrated into the host cell genome [26, 27]. Earlier studies in our lab have focused on providing a complete kinetic and thermodynamic understanding of catalytic activities of HIV-1 RT in the elongation steps involving RNA-dependent and DNA-dependent DNA polymerization as well as RNase H cleavage. Other studies have examined the template switching and strand transfer reactions [28-34]. There is much less kinetic and mechanistic information available concerning the crucial initiation process in which human tRNA3Lys binds to a region on the RNA genome known as the primer binding site (PBS) and initiates DNA synthesis. Accordingly, during the previous and current periods, one of our objectives has been to examine the reaction kinetics and develop an understanding of the mechanistic details of this tRNA initiation step. These studies were conducted using a model RNA/RNA primer-template, full length synthetic tRNA3Lys, and natural human tRNA3Lys as a primer. Enzymatic and chemical probing studies have suggested that the formation of the HIV-1 RNA/tRNA3Lys complex is accompanied by numerous intra- and inter-molecular interactions that result in a highly order structure [35-38]. Interestingly, these interactions require post-transcriptional modifications of tRNALys and accordingly, are lacking in studies with synthetic tRNA3Lys [35, 36, 39, 40].
Role of the Nucleocapsid Protein in HIV RT:
Along with the duplicate copies of the HIV RNA genome, infectious virions contain large amounts of nucleocapsid proteins that are believed to modulate various enzyme activities during the replication of the RNA genome. The two HIV nucleocapsid (NC) proteins receiving the most study are NC p15 and a shortened version NC p7 [41]. These NC proteins contain two zinc finger binding motifs and the structure of NCp7 has been determined [42-44]. The role of NCp7 in HIV-1 viral replication is still not well defined but may be involved in dimerization, packaging of genomic RNA, viral morphogenesis, and the reverse transcription process for RT [45-50]. Thus, in vitro, NCp7 has been shown to (1) promote annealing of the tRNA3Lys to the PBS [51-53]; (2) accelerate (−) strand DNA transfer during proviral DNA synthesis [32, 51, 54, 55], and (3) enhance processivity and RNase H activity of RT [29, 31, 54, 56, 57]. A direct interaction between NCp7 and RT has been hypothesized and recently demonstrated in vitro [58]. These studies are primarily descriptive in nature and the mechanistic basis of these interactions and the effects of NCp7 on the reaction kinetics of RT have not been examined in detail. Understanding functions of the NC proteins in facilitating replication of the HIV viral genome may provide important insights into potential targets for antiviral chemotherapeutic intervention.
Previous work to develop non-cleavable heterodimers as bifunctional inhibitors has met with limited success and the evidence to date indicates that a true bifunctional two-site inhibitor such as those of the present invention has yet to be realized. To date, all these studies have used cell-based assays to evaluate the efficacy of the bifunctional nucleoside inhibitors, and it is likely that cellular phosphorylation of the bifunctional nucleoside to the triphosphate is a prerequisite for optimal NRTI activity. Therefore, it is unclear whether the lack of activity in earlier reports of bifunctional inhibitors was due to cellular factors such as a failure to be phosphorylated by nucleoside/nucleotide kinases or at the enzyme level due to lack of binding and inhibition of RT.